For both clinical therapy and biomedical research applications, there is a great need for a rapid and efficient technique for the separation and isolation of specific cell types from a mixture. For example, in the treatment of leukemia, cancerous lymphocytes can be separated from erythrocytes and other cell types in the blood. The healthy blood can then be transfused back into the patient's body. For treatment of childhood bone marrow cancer, diseased cells can be separated in vitro, and the normal cells can be put back into the body, avoiding the laborious (and often futile) process of looking for appropriate donors for a transplant. In AIDS patients, helper T cells which harbor the HIV virus within its genome need to be selectively removed, leaving behind B cells, erythrocytes and other healthy cells.
The use of magnetic particles for binding and separating target cells is known, see U.S. Pat. No. 4,965,007. In one technique, micron sized latex spheres with magnetic cores are coated with secondary antibodies that are specific to the target cells. The antibody bound target cells then bind to the microspheres and are separated from the mixture by a magnetic field. This method requires two antibodies and relies on three successful bindings--between latex/antibody, antibody/antibody and antibody/ target. For maximum efficiency, the antibodies on the latex should have their Fab regions exposed, a feature that has been particularly difficult to ensure. In addition, a new secondary antibody must be coated on the microspheres each time a new cell is targeted for removal. Furthermore, simultaneous removal of more than a single target from a mixture requires successful coating of multiple secondary antibodies on the microspheres.
An alternative method consists of coating functionalized silane layers on micron-sized magnetic particles. Ligands can then attach to the exposed functional group. Ligand-antiligand attraction and binding followed by exposure to a magnetic field gradient allows the antiligand to be preferentially separated. The ligand can be an antibody to a target cell, while the antiligand can be the target material.
The invention disclosed in the parent application was directed to an efficient and rapid method for sorting specific cell types from a mixture by using Fc receptor coated magnetic vesicles (liposomes). The invention was also useful for the separation of high valued biological macromolecules that appear in extremely low concentrations.
Briefly, the invention of the parent application comprised forming compartmented vesicles, preferably single compartment vesicles, having magnetic particles encapsulated therein. Embodied in the vesicle wall were Fc receptor proteins. The Fc domain of an antibody bound to the Fc site of the protein and the antibody bound to the surface antigen of a target cell. This complex of magnetic vesicle/Fc receptor/antibody/target cells was removed and each vesicle antibody-target cell was independently recovered.
The present invention deals with bioseparations using a ferritin-protein A conjugate. Ferritin, an iron storage protein, was disclosed in the parent application for a different purpose. There, the electron dense nature of ferritin was exploited to visualize by electron microscopy the location of protein-A on the vesicle walls.
In the present invention, we exploit the magnetic susceptibility of ferritin, rather than its electron dense property to effect a separation. Broadly, in the present invention, commercially available ferritin-protein A conjugates (Sigma Immunochemicals) are mixed into a solution containing antibody bound target cells. Because protein-A is an Fc receptor, the ferritin-protein-A conjugate binds to the exposed Fc domains of the antibodies, creating ferritinprotein A-antibody-target cell complexes. A ferrofluid containing a stable suspension of magnetic nanoparticles is then added to the solution. Because of the high magnetic field gradients of the ferrofluid particles, they atttach to the magnetically susceptible ferritin portion of the complex. The ferrofluid particle-target cell complex is then removed from solution by exposure to magnetized steel wool .